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(Hypertension. 2003;41:604.)
© 2003 American Heart Association, Inc.

Scientific Contributions

G₁₂- and G₁₃-Protein Subunit Linkage of D₅ Dopamine Receptors in the Nephron

Shaopeng Zheng; Peiying Yu; Chunyu Zeng; Zheng Wang; Zhiwei Yang; Peter M. Andrews; Robin A. Felder; Pedro A. Jose

From the Departments of Pediatrics (S.Z., P.Y., C.Z., Z.W., Z.Y., P.A.J.), Physiology and Biophysics (Z.Y., P.A.J.), and Cell Biology (P.M.A.), Georgetown University Medical Center, Washington, DC, and the Department of Pathology, The University of Virginia Health Sciences Center (R.A.F.), Charlottesville, Va.

Correspondence to Dr Pedro A. Jose, Georgetown University Medical Center, 3800 Reservoir Road, NW, Washington, DC 20007. E-mail pjose01{at}georgetown.edu

	Abstract

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The roles of the G-protein -subunits, G_s, G_i, and G_q/11, in the signal transduction of the D₁-like dopamine receptors, D₁ and D₅, have been deciphered. G₁₂ and G₁₃, members of the 4th family of G protein subunits, are not linked with D₁ receptors, and their linkage to D₅ receptors is not known. Therefore, we studied the expression of G₁₂ and G₁₃ and interaction with D₅ dopamine receptors in the kidney from normotensive Wistar-Kyoto (WKY) rats and D₅ receptor-transfected HEK293 cells. G₁₂ and G₁₃ were found in the proximal tubule, distal convoluted tubule, and artery and vein in the WKY rat kidney. Whereas G₁₂ was expressed in the ascending limb of Henle, G₁₃ was expressed in the collecting duct and juxtaglomerular cells. In renal proximal tubules, G₁₂ and G₁₃, as with D₅ receptors, were expressed in brush border membranes. Laser confocal microscopy revealed the colocalization of D₅ receptors with G₁₂ and G₁₃ in rat renal brush border membranes, immortalized rat renal proximal tubule cells, and D₅ receptor-transfected HEK293 cells. In these cells, a D₁-like agonist, fenoldopam, increased the association of G₁₂ and G₁₃ with D₅ receptors, results that were corroborated by immunoprecipitation experiments. We conclude that although both D₁ and D₅ receptors are linked to Gs, they are differentially linked to G₁₂ and G₁₃. The consequences of the differential G-protein subunit linkage on D₁- and D₅-mediated sodium transport remains to be determined.

Key Words: receptors, dopamine • G proteins • juxtaglomerular apparatus • immunohistochemistry

	Introduction

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Dopamine is an endogenous neurotransmitter catecholamine that also serves as a biochemical precursor of norepinephrine and epinephrine in neural tissue. Dopamine can be synthesized in nonneural tissues as well; however, in this setting, dopamine is not converted to norepinephrine. It is now recognized that dopamine, produced in nonneural sites, functions in an autocrine or paracrine manner and serves an important role in the regulation of sodium balance by direct actions on renal and intestinal epithelial ion transport, by interaction with other receptors, and by modulation of the secretion of hormonal/humoral agents such as aldosterone, catecholamines, renin, and vasopressin.¹ Dopamine additionally affects salt and water balance by acting on brain appetite centers. The actions of dopamine on water and electrolyte transport, which are present but modest in euvolemic conditions, become magnified during moderate sodium excess. Thus, after a moderate acute or chronic sodium load, more than 50% of sodium excretion is under the control of dopamine produced by the renal proximal tubule.^1,2

The effects of dopamine are exerted by cell surface receptors that belong to the rhodopsin-like or class A family of membrane receptors.³ These receptors, characterized by 7-membrane spanning domains, are called G-protein-coupled receptors because of their interaction with heterotrimeric G proteins, composed of -, ß-, and -subunits.^3,4 There are more than 20 G-subunits, grouped into 4 subfamilies (G_S, G_i, G_q, and G₁₂). There are 5 Gß-subunits, grouped into 2 subfamilies, and 12 G-subunits, grouped into 4 subfamilies. In mammals, the 2 D₁-like dopamine receptors, D₁ and D₅, are coupled to the stimulatory G-subunit, G_S, whereas the 3 D₂-like receptors, D₂, D₃, and D₄, are coupled to the inhibitory G-subunit, G_i; G_S is stimulatory, whereas G_i is inhibitory of adenylyl cyclase activity.^1,2,5,6 Both D₁ and D₅ receptors can also couple to G_15/16 but not to G₁₄.⁷ Under certain circumstances, for example, in the presence of pertussis toxin, the D₁ but not the D₅ receptor can couple to any of the 3 isoforms of G_i (G_i-1, G_i-2, and G_i-3).^8–10 The D₁ receptor but not the D₅ receptor also couples to Gq and stimulates phospholipase Cß in the presence of the adaptor protein calcyon.^11–14 Thus, the roles of G-protein -subunits G_s, G_i, and G_q/11 in the signal transduction of the D₁-like dopamine receptors D₁ and D₅ have been deciphered. G₁₂ and G₁₃, members of the 4th family of G protein subunits, are not linked with D₁ receptors, and their linkage to D₅ receptors is not known.^15,16 Therefore, we studied the expression of G₁₂ and G₁₃ in the kidney from the normotensive Wistar-Kyoto (WKY) rat, immortalized renal proximal tubule cells, and in HEK293 cells expressing D₅ receptors.

	Methods

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Preparation of Renal Brush Border Membranes
Kidneys were obtained from pentobarbital-anesthetized (50 mg/kg body wt, by intraperitoneal injection) WKY rats (9 to 16 weeks old). Brush border membranes (BBMs) were prepared by MnCl₂ precipitation and differential centrifugation¹⁷ and studied under approved protocols with institutional guidelines. The BBMs have no immunoblottable sodium-hydrogen exchanger 1 (NHE1) (marker for basolateral membranes) but express immunoreactive NHE3 (marker for BBMs), indicating minimal contamination with basolateral membranes.¹⁸ Protein concentrations were determined by Detergent Compatible Protein Assay Kit (Bio-Rad).

Immunoblotting
Immunoblotting was performed as reported,^10,19 with polyclonal rabbit anti-human G₁₂, rabbit anti-mouse G₁₃ (Santa Cruz Biotechnology, Inc),^20,21 or rabbit anti-human D₅ receptor (Research Genetics)²² diluted 1:200 to 500 in blocking solution for 1 hour at room temperature. The immunoblots were visualized and quantified as reported.^10,19,23 Controls included antibody preadsorbed with immunizing peptide and preimmune serum substituted for the antibody or antiserum.

Immunoprecipitation Studies
BBMs were treated with vehicle (dd H₂O) or fenoldopam (5x10^-6 mol/L) for 30 minutes at room temperature and then lysed with lysis buffer (50 mmol/L Tris-Cl, pH, 7.4, 1% NP-40, 0.1% SDS, 1 mmol/L phenylmethysulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin) on ice for 1 hour.^10,23 After centrifugation at 16 000g at 4°C, the lysates (supernatants) were precleared by adding 0.2 µg of normal rabbit IgG per 800 µg of protein and 20 µL of protein G agarose (Santa Cruz Biotechnology, Inc) for 1 hour and recentrifuged at 16, 000g for 2 minutes. The precleared lysates (1 mL) were then incubated with 1 µg of rabbit anti-human D₅ dopamine receptor antibody and 20 µL of protein G agarose with rocking overnight at 4°C. The samples were pelleted by centrifugation at 16, 000g for 2 minutes at 4°C, washed 3 times with lysis buffer, and resuspended in 25 µL of electrophoresis loading buffer. The samples were boiled at 100°C in a water bath for 10 minutes, subjected to 8% SDS-PAGE, and immunoblotted as above.

HEK293 cells were transfected with human D₅ receptor fused to a V5-His tag at the C-terminus or empty vector (pcDNA6/V5-His), which served as a control. The cells were incubated with vehicle or fenoldopam (5x10^-6 mol/L) for 30 minutes. The cells (80% confluence) were extracted in ice-cold lysis buffer (PBS with 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin), sonicated, kept on ice for 1 hour, and centrifuged at 16, 000g for 30 minutes. The supernatants were stored at -70°C until use for immunoprecipitation. Equal amounts of lysates were incubated with affinity-purified anti-D₅ receptor antibody (1 µg/mL) for 1 hour and protein-G agarose at 4°C for 12 hours. The immunoprecipitates were pelleted and washed 4 times with lysis buffer. The pellets were suspended in sample buffer, boiled for 10 minutes, and subjected to immunoblotting with the G₁₂ or G₁₃ receptor antibody. To determine the specificity of the bands, normal rabbit IgG (negative control) and G₁₂ or G₁₃ receptor antibody (positive control) were used as immunoprecipitants instead of the D₅ receptor antibody.

Immunohistochemistry
The rat kidneys were cleared of blood with oxygenated saline and kept in Histochoice (Amresco)¹⁸ for 1 to 2 days at 4°C. The samples were embedded in paraffin, and 4-µm sections were mounted on slides. After deparaffination, rehydration, permeabilization, and blocking (5% normal goat serum in PBS), the slides were subjected to immunostaining with primary antibodies used above (1:100 to 200 in blocking solution) at 4°C overnight. Biotinylated anti-rabbit-IgG and diaminobenzidine detection system (Vectastain ABC Kit, Vector Labs) were used for color development. The samples were counterstained with hematoxylin. Antibody specificity was determined using controls as listed above, including incubation without the primary antibodies.

Immunofluorescence Staining
Immortalized Renal Proximal Tubule and HEK293 Cells
HEK293 cells (ATCC) and immortalized renal proximal tubule cells from normotensive WKY rats were prepared as described.^10,23 On achieving 50% confluence in coverslips, the cells were treated with vehicle (dd H₂O) or fenoldopam (5x10^-6 mol/L) for 30 minutes at 37°C and fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature.

Kidneys
The kidneys were perfused with saline as described above and flash-frozen immediately in Optimal Cutting Temperature Compound. The frozen kidney sections, mounted on slides, were fixed with cold methanol for 10 minutes.

The renal proximal tubule cells or frozen rat kidney sections were washed with PBS and permeabilized (0.1% Triton X-100 in PBS) for 30 minutes at room temperature, blocked (5% normal goat serum in PBS), and stained with the G₁₂ or G₁₃ antibodies. Visualization was accomplished with fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (Jackson Laboratory), blocking of the secondary antibody with rabbit IgG (25 µg/mL), and, finally, staining with rabbit anti-human D₅ antibody conjugated to Alexa 568.

The colocalization of G₁₂ and G₁₃ with the D₅ receptor was further studied in HEK293 cells transfected with the D₅ receptor cDNA. When anti-G₁₂ and anti-G₁₃ antibodies were used as the primary antibodies, the secondary anti-rabbit antibody was labeled with Texas red. Fluorescein isothiocyanate-conjugated anti-mouse secondary antibody was used for the HEK293 D₅ receptor immunostaining. The slides or coverslips were washed and mounted for fluorescence studies (Vectashield, H-1000, Vector Laboratories). Negative controls included absence of the primary or the secondary antibody. The images were examined with an Olympus AX70 laser confocal microscope with a x60 objective at an excitation wavelength of 480 and 560 nm; emission was at 535 and 645 nm, respectively.

Statistical Analysis
The data are expressed as mean±SEM. Differences among means were determined by the Student t test (n=2) or ANOVA (n >2) followed by the Duncan test; a probability value <0.05 was considered as significant.

	Results

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Immunohistochemistry of G₁₂, G₁₃, and D₅
G₁₂ and G₁₃ were found in the proximal tubules as well as the arteries and veins in the WKY rat kidney (Figures 1A and 1B). In the proximal tubule, G₁₂ was expressed predominantly in BBMs and subjacent areas, whereas the G₁₃ was predominantly expressed subjacent to the BBMs. Whereas G₁₂ was expressed in the ascending limb of Henle and cortical collecting duct, G₁₃ was expressed in the distal convoluted tubule, the medullary collecting duct, and the juxtaglomerular cell (Figure 1B). The D₅ receptor was in the proximal tubule, and especially at the BBMs (Figure 1C) similar to those noted with G₁₂ and G₁₃ (Figures 1A and 1B). No staining was seen when the antibodies were preadsorbed with the immunizing peptide (Figures 1A and 1B), when preimmune serum was used (Figure 1C), or when the primary or secondary antibodies were omitted (not shown).

View larger version (87K): [in this window] [in a new window]   Figure 1. Immunohistochemical staining of rat kidneys. A, G12 is located in renal proximal tubules (PT), especially at the brush border and subjacent areas, ascending limbs of Henle, cortical collecting ducts, arteries, and veins. No staining is seen when G12 antibody is preadsorbed with immunizing peptide control peptide (CP). These studies were performed at least 3 times. B, G13 is located in renal proximal tubules, especially subjacent to the brush border, distal convoluted tubules, medullary collecting ducts, juxtaglomerular (JG) cells, arteries, and veins. There is negligible staining in the glomerulus. No staining is seen when G13 antibody is preadsorbed with the immunizing peptide CP. These studies were performed at least 3 times. C, D5 receptors expressed in proximal tubule brush borders. No staining is noted in preimmune serum and when the D5-receptor antibodies are preadsorbed with immunizing peptide (not shown). These studies were performed at least 3 times.

Immunoblotting of G₁₂, G₁₃, D₁, and D₅ Receptors
Because of the apparently strong expression of G₁₂, G₁₃, and D₅ receptors (Figures 1A, 1B, and 1C) and the other D₁-like receptor, the D₁ receptor,^23–26 in proximal tubule BBMs, we next assessed the expression of these proteins in renal BBMs. The major specific bands for G₁₂, G₁₃, (43 kDa), and D₅ receptors (50 kDa) were detected in BBMs, confirming the immunohistochemistry data. As with the immunohistochemistry studies, preincubation with their respective immunizing peptides eliminated the immunoblots (Figures 2A through 2C).

View larger version (18K): [in this window] [in a new window]   Figure 2. Immunoblotting of G12, G13, and D5 receptors in renal BBMs. Renal BBMs were prepared by differential centrifugation and MnCl2 precipitation17 and immunoblotted for G12 (A), G13 (B), and D5 (C). Striatum was used for positive control (A and B). No bands were seen when antibodies were preadsorbed with their respective immunizing peptides. These studies were performed at least 3 times. Molecular sizes are shown.

Laser Confocal Microscopy
We next determined whether G₁₂ or G₁₃ colocalized with the D₅ receptor. The anti-G₁₂ and anti-G₁₃ antibodies were indirectly labeled with fluorescein isothiocyanate, whereas the anti-D₅ receptor antibodies were directly labeled with Texas Red. Laser confocal microscopy of flash-frozen kidneys showed that the fluorescence staining of G₁₂, G₁₃, and D₅ is mainly located and colocalized at the BBMs and subjacent areas (Figure 3A); there was minimal autofluorescence, and no fluorescence was seen in the absence of the antibodies (not shown). To determine whether D₁-like agonist stimulation can enhance the colocalization of these proteins, the laser confocal microscopy studies were repeated in immortalized rat renal proximal tubule cells. Fenoldopam, a D₁-like agonist (5x10^-6 mol/L, 30 minutes), appeared to increase the colocalization of D₅ receptors with G₁₂ and G₁₃ (Figures 3B and 3C).

View larger version (69K): [in this window] [in a new window]   Figure 3. Laser confocal microscopy of G12, G13, and D5 receptors. Flash-frozen sections of kidneys were stained for G12 or G13 (green) or D5 receptors (red); fluorescence images were merged (yellow). No autofluorescence was seen (negative control, not shown) (A). Colocalization of G12 or G13 (green) and D5 receptors (red) was further examined in rat renal proximal tubule cells (B and C) and HEK293 cells (D and E). Fenoldopam (5x10-6 mol/L, 30 minutes) appeared to increase their colocalization (yellow) (B, C, D, and E). No colocalization was noted in HEK293 cells transfected with empty vector (not shown).

HEK293 Cells
HEK293 cells have been reported to express G₁₂.^27,28 HEK293 cells also express G₁₃,²⁷ although to a lesser degree than G₁₂, which may have led earlier reports to conclude that G₁₃ is not endogenously expressed in this cell line.²⁸ Like the results observed with renal proximal tubule cells, D₅ colocalized with G₁₂, and G₁₃, which was also increased by fenoldopam (Figures 3D and 3E). No such colocalization was found in HEK293 cells transfected with the empty vector (not shown).

Immunoprecipitation of G₁₂, G₁₃, and D₅ Receptors
To confirm the apparent interaction between G₁₂ or G₁₃ with D₅ receptors noted in the laser confocal microscopic studies, we determined whether G₁₂ or G₁₃ coimmunoprecipitated with D₅ receptors in BBMs because of the dense expression of these proteins at this site and in HEK293 cells. G₁₂ and G₁₃ coimmunoprecipitated with D₅ receptors in BBM (Figures 4A and 4B) and HEK293 cells (Figures 4C and 4D), which were increased after D₁-like agonist stimulation (fenoldopam, 5x10^-6 mol/L, 30 minutes). There was negligible coimmunoprecipitation when the immunoprecipitant was IgG instead of anti-D₅ receptor antibodies and when the vector-transfected HEK293 cell lysates were used. Similar results were obtained when anti-V5 antibodies were used; immunoprecipitation and anti-G₁₂ and anti-G₁₃ antibodies were used for immunoblotting (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 4. Coimmunoprecipitation of G12 or G13 and D5 receptors. Renal BBMs17 and HEK293 cells were treated with vehicle or fenoldopam (F) (5x10-6 mol/L) for 30 minutes. The same amount of proteins was then immunoprecipitated with D5 receptor antibody and immunoblotted for G12 (A, BBM; C, HEK293) or G13 (B, BBM; D, HEK293). Negligible coimmunoprecipitation occurred with rat IgG as the immunoprecipitant. For positive controls, anti-G12 and G13 antibodies (1 µg/mL) were used as immunoprecipitants; for negative control, normal rabbit IgG (1 µg/mL) was used as immunoprecipitant instead of anti-D5 antibodies and immunoblotted with anti-G12 or G13 antibodies as above. P indicates positive control. *P<0.05 vs control (C) (n=5 to 6), t test.

	Discussion

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These studies describe the distribution of G₁₂ and G₁₃ in the rat kidney. Although both members of the 4th class of G-subunits are found in proximal tubules, arteries, and veins, G₁₂ is expressed in the ascending limbs of Henle and cortical collecting ducts, whereas G₁₃ is expressed in the medullary collecting ducts. Both G₁₂ and G₁₂ are well expressed in BBMs, the area in the proximal tubule where D₁ and D₅ dopamine receptors are also well expressed.^23–26

In the current report, we find linkage of D₅ receptors to G₁₂ and G₁₃. The linkage was found in renal proximal tubules in native kidneys, in immortalized renal proximal tubule cells, and in HEK293 cells heterologously expressing the D₅ receptor. This is in contrast to that absence of linkage of the D₁ receptor to either G₁₂ or G₁₃.¹⁵ G₁₂ and G₁₃ and D₁-like receptors have been reported to influence sodium transport by regulating the activity of the sodium/hydrogen exchanger, type 3 (NHE3),^1,2,29 the major transporter of sodium in the renal proximal tubular BBMs.^10,30–32 While G₁₂ and G₁₃ stimulate NHE3 activity,²⁹ D₁-like receptors inhibit NHE3 activity through G_S.^10,30–32 It is not known, however, whether both D₁-like receptors, D₁ or D₅ or only one of them, inhibits NHE3 activity in renal proximal tubules. The fact that the D₁-like agonist fenoldopam increases the interaction between the D₅ receptor with either G₁₂ and G₁₃ in BBMs suggests that the D₅ receptor may participate in the regulation of NHE3 activity. Interestingly, the amount of cAMP produced in response to D₁-like agonist stimulation by D₁ receptors is greater than that caused by D₅ receptors.²⁶ Because the inhibition of NHE3 activity by D₁-like agonists is caused to large extent by activation of adenylyl cyclase activity, one may assume that the inhibition of NHE3 activity by D₁-like receptors is due mainly to the D₁ receptor. However, D₁-like receptors can inhibit NHE3 activity independent of cAMP,³³ and G₁₃ has been reported to activate protein kinase A, independent of cAMP production.³⁴ Because binding of G₁₂ and G₁₃ to D₅ receptors increase with D₁-like agonist stimulation, it is conceivable that this could lead to a decrease in the amount of G₁₂ and G₁₃ that can bind to and stimulate NHE3 activity, resulting in its inhibition.

One interesting observation in this report is the expression of G₁₃ in the juxtaglomerular cell, where renin is produced. Both D₁ and D₅ receptors are expressed in the juxtaglomerular cells, and D₁ receptors stimulate renin secretion.^35,36 However, as stated above, the D₁ receptor is not linked to G₁₃. In a preliminary report, we found that the D₃ receptor is linked to G₁₃³⁷; the D₃ receptor negatively regulates renin secretion.^38,39 G₁₃ can increase intracellular calcium and calcium can decrease renin secretion.^40,41 It is tempting to speculate that G₁₃ may be important in the negative regulation of renin secretion.

In summary, we found nephron segment-specific expression of G₁₂ and G₁₃, especially in renal BBMs. The D₅ but not the D₁ receptor colocalizes and interacts with both G subtypes, the interaction of which is increased by D₁-like agonist stimulation. However, the role of G₁₂ and G₁₃ in the regulation of D₅ receptor function remains to be determined.

Perspectives
G₁₂ and G₁₃ may participate in the regulation of D₅ receptor function. G₁₂ stimulation of NHE activity has been related to phospholipase D.⁴² Because D₅ receptors inhibit phospholipase D activity (unpublished observations), the G₁₂/phospholipase D pathway by be the mechanism by which D₁-like receptors inhibit renal proximal tubular sodium transport independent of PKA. G₁₃ in juxtaglomerular cells could participate in the regulation of renin secretion.

	Acknowledgments

 
These studies were supported in part by grants from the National Institutes of Health (HL 23081, DK 39308, HL68686, DK52612, and HL 62211).

Received November 18, 2002; first decision December 12, 2002; accepted January 9, 2003.

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